Copolymer of l-leucine and dl-phenylalanine



Patented Nov. 3, 1953 COPOLYMER OF L-LEUCINE AND DL-PHENYLALANINE Robert B. Woodward, Cambridge, Mass.

N Drawing. Application September 6, 1952, Serial No. 308,303

7 Claims.

This invention relates to synthetic protein analogues and more particularly to new synthetic linear polypeptides, that is, polyamides of a amino acids and to the process for preparing same. More particularly the novel compositions of this invention are the copolymers of the a-amino acids L-leucine and DL-phenylalanine. This class of copolymers has the general structural formula 0 H H liralfl L I l. (1) wherein Z is the non-active-hydrogen moiety of the initiator, R is selected from the group consisting of benzyl and isopropyl radicals, each in material quantities, and n is an integer of at least 100.

One object of the present invention is the provision of a novel process analogous to polymerization for preparing high molecular weight compounds of the above type, particularly for the preparation of novel polypeptides having molecular weights sufiiciently high to permit the formation of films or sheets and fibers. Molecular weights in excess of 13,000 and, in fact, molecular weights up to 100,000 and higher are readily obtained.

Another object of the invention is the produc-- tion of fibers and films from the novel copolymer compositions of this invention.

According to the present invention, polypeptides of almost any desired molecular weight can be prepared by a self-propagating chain reaction analogous to polymerization. The components for carrying out the preparation of polypeptides in this manner are:

I. An initiator which can be any donor molecule, i. e., any molecule which has one or more atoms bearing unshared electron pairs such, for example, as a substance ZH in which H is an active hydrogen atom; that is, a substance such as water, an alcohol, a primary or secondary amine, an amino acid or a mercaptan.

II. A monomer of the general formula R-CH-NH o=o c=o o wherein R. is as indicated in (1).

When these two components are introduced into a suitable solvent, reaction ensues in the following manner:

(a) One molecule of initiator attacks one molecule of monomer:

+ O: /C=O R R OH (d) Like (3), (5) is inherently unstable and r 1 I I decomposes in a similar manner, liberating again be made. The length of the chain depends on' (a) the supply of monomer molecules, (b) the relative concentrations of monomer and initiator molecules, and (c) the relative rates of the chain-propagating and chain-initiating steps.

The processpossesses many advantages as a model of polymerization reactions in that chainterminating, chain-transfer, and, very probably, As in chain-branching reactions, are absent. other polymerization reactions, the products formed by the reaction in its'simplest forms are ordinarily mixtures of molecules-of the general formula (1) with difierent "values of The simplicity of the system, however, permits a greater degree of control over the molecular weight distribution than is ordinarily possible in previously known polymerization-reactions."

Thus, if an initiator is usedwhichreacts rela assis /2 anhydrides, esters and acid chlorides. For ex ample, acetic anhydride will react with the active centers of the growing chains in the sense;

---NHz+(CH.7CO)2O- A h NH--CO-CH:4+C'I-I$C'OOH ('7) It is further significant that any product bf the present invention can be used as the hi tiator of a further chain reaction of the abbve type. Thus, any substance of the general formula (1) with a given value of n can be converted into a similar substance with a larger value of '11 simply by further reaction with a new supply of monomer molecules.

in a similar way, other large molecules of natural or synthetic origin can be'used as the initiator. In this fashion, for example, natural proteins can be modified by theaddition of polypeptide chains at any point where the original moiecuie contains free-N112, -UH, --SH, 01 si'milar groups. Likewise, a synthetic polymer, such as polyvinyl alcohol, can be modified by the addition of polypeptide chains at various of its -QH group's, viz:

om-011a. (wa es- H (COCHNH),.H

One of the important aspects of the process of the present invention is that it makes possible the formation of polypeptides of any desired moleculai' weight wherein there can be obtained repeating units of somewhat different chemical configuration; that is, high molecular weight compounds analogous to copolymers. For. example, it is possible to take various mixtures of the N-carboxy amino acid anhydrides such as N-carboxy-L-leucine anhydride, N-carboxy-DL- phenylalanine 'anh'ydride, N carboxy-Dh-waline anhydride, O-acetyl-N-carboXy-L-tyrosine anhydride et-N-cafbomethoxym-Necarboxy-DL-lysine anhydride, O-carbomethoXy-N-carboxy-L-tyrosine anhydride, etc., and by. dissolving the same iii'a suitable solvent in the presence of an initiator to carry out 'theiorination of a high iiioleulai weight polypeptide whose -M groiip's will be variously the corresponding 'g'foiips'of the said monomeric materials.

It is to be noted from the foregoing'that. the

polypeptides of the present invention contain as.

their end groups a radicalzderived from the :initi ator molecule and by a suitable selection of initi ators various properties can be impartedqtothei. polypeptides of the; invention adapting them for.

4 of methyl chloroformate added in small portions, with shaking after each addition. An 8.2-g. portion of anhydrous sodium carbonate was then added. During the reaction a lumpy precipitate was formed. After shaking for one-half hour a clear solution was obtained. After standing overnight, the solution was acidified to pH 3 which deposited the reaction product, N-carbomethoxy-L-leucine, as an oil. The mixture was extracted with three portions of diethyl ether, and the ethereal solution filtered and evaporated "ujnar vaeuum to yield the nearly colorless, oily N carbomethoxy-L-leucine.

- ---The N-carbomethoxy-L-leucine was converted to N-carbomethoxy-L-leucine acid chloride by heating "with 32 cc. of thionyl chloride for one example, for medicinal and pharmaceutical uses;

The following examples are illustrative of the invention. i

' Esaiiipl" 1 As'oliitio'r'i prepar d cooled too c.111- afi iceiiatn and nag. (recs-c.)

hour at 40 C. in a water bath. A reaction occarried with evolution of hydrogen chloride gas.

xcess thionyl chloride was then removed by heating under vacuum at 40 C. The anhydri'de was obtained by further heating. The reaction began at about C. with evolution of methyl. chloride and was complete after heating at (3.. After standing overnight therefor one hour. action miXture deposited yellow brown crystals of crude N-carboxy L leucine anhydride. Crystallization from a diethyl ether-petroleum ether mixture gave in two crops 15;? g. of tan crystals of the anhydride. This was taken up in boiling cyclohexane, decolor'ized with charcoal, and gave on two crystallizatidns 8.10 g. (yield 34 per cent based on L-leucinehof nearly colorless needles of pure N-carboXy-L-leucine anhydride, melt ing point YB-78 C (timbre) 1 =+3e75 (0.5000 g. in 25 cc. benzene).

Calculated ror CtHiiON; N, 8.91. Found? c, 53.70: H, 7.01; N, 8.70.

Formula:

(crimcncni en r rn I 'o'=" ,c='o

Example 2 To a solution of 26.0 g. or ion-phenylal nine in 12 0 cc. "of 1 N so diilinhydroiiide was added 11.0 cc. bfhithYl chlofdfoiniate and 6.4 g. 6f all: hydrous sodium, carbonate. The" reaction mix- ,ture'was came-r1 in ce bath and shaken dcethereal solution evaporated under vacuum to give N-carbomethpxy-Dl i phenylalanine as a colorle'ss glassy solid. This was heated with 3066.

of thionyl chloride at 40 c. for forty-five, mire utes, than t 0 c. or firtejen minutes un er vacuum to give a massof bright yellowcry'stals,

To these crystals 10 cc. ofb'iizene 'wa's'fadded and t hemixtur' a'gainheated 511, 0" 0. rorfifte xiii minutes. The residue was crystallized from ten zerie and gave' in twqcrdps 16.4 "g7. ('71 er cent, yield) of colorless plates of'N=c'arboXy'-DL -phenylalanine anhydride.. A sample wasj'fur'ther purified by another crystallization from benzene to give pure N-carboxyeDL-phenylalanine anhydride, melting point 127-1275 C. (corn).

Calculated for CwI-IeOaN: C, 62,82; H, 4.47; N,

7.32. Found: c, 62290; H, 4.83; N; 7.35:

Example 3 One gram each of N-carboxy-L-leucine anhydride and N-carboxy-DL-phenylalanine anhydride were dissolved in approximately '75 cc. of ordinary reagent benzene (ACS reagent grade benzene) and sufficient acetone to bring the less soluble phenylalanine derivative completely into solution. The very small amount of water pres ent in the reagent grade benzene served as initiator. The solution was allowed to stand at room temperature (approximately 0.). As the reaction proceeded, the solution became more and more viscous. When, after two weeks, portions of the solution were cast on glass, optically clear, mechanically stable, tough films were readily formed. Thus, the film was made up of molecules of the structure (1) With'high values of n. Chemical analysis for the a-amino nitrogen by the Van Slyke method has shown that n is at least 100.

Example 4 A 2 per cent benzene solution of the polypeptide obtained in accordance with the process of Example 3 was introduced into a hypodermic syringe and ejected therefrom into petroleum ether, acetone and warm air, producing thin filaments or fibers of said polypeptide in each of said fluids. Other suitable mediums which can be employed are hexane, ethyl alcohol, mixtures thereof and the like.

The polypeptide copolymer of L-leucine and DL-phenylalanine Was also found to produce an excellent film when cast in a thin sheet from benzene solution.

The initiator containing the active hydrogen atom should preferably be present in an amount less than one per cent by weight, based on the N-carboxyanhydride reaction mixture. Thus ACS reagent grade benzene has an upper limit for water of about 0.02 per cent. Accordingly, it is seen that the limit of the water initiator present in Example 3 is about 0.66 per cent by weight, based on the N-carboxyanhydride reaction mixture. Smaller quantities of initiator are desirable in obtaining copolymer compositions of high molecular weight. It has also been found that careful purification of the N-carboxyanhydride is a necessity for obtaining high molecular weight polypeptide compositions.

As pointed out hereinabove, the length of the polypeptide chains obtained in accordance with the present invention depends in part on the relative rates of the chain-propagating and chain-initiating processes. Water acts as an initiator whose initiating reaction, in general, is substantially slower than the chain-propagating reaction, as evidenced by the fact that the chain propagation may take place when carried out in a water solution. Examples of compositions whose initiating reaction is more rapid than the chain propagation are those containing hydroxyl ions, for example, solutions of quaternary ammonium hydroxides such as Triton B, which is a 40% aqueous solution of benzyl trimethyl ammonium hydroxide, and solutions of alkali hydroxides, such as sodium hydroxide and potassium hydroxide. Amino acids are donor molecules which act to initiate the reaction at approximately the same rate as the rate of propagation or chain growth.

Whereas Example 3 shows the preparation of the polypeptide copolymer at about room temperature, it is apparent that other temperaturetime conditions can be employed, for example,

heating the inert organic solvent containing the reaction mixture to a temperature of about C. for a substantially shorter period of time than when the polymerization is brought about at room temperature. Other suitabl reaction conditions are apparent to the skilled chemist.

The term linear polypeptide copolymer as used in the claims is to be understood to refer to a polypeptide having repeating units of the general configuration as shown at (1), which repeating units appear in the polypeptide chain one hundred times or more, that is, the valve 11. is or greater. Thus, the novel polypeptide copolymers of this invention have a molecular weight of at least about 13,000, where the said copolymer consists of substantially equimolecular quantities of the monomers L-leucine and DL-phenylalanine.

Synthetic polypeptides of low molecular weight have been reported but these have been either the undesired products of reactions intended for other purposes (Leuchs and Geiger, Ben, 41, 1721 (1908) Curtius and Sieber, Ben, 55, 1543 (1922) Wessely and John, Z. physiol. Chem., 38 (1927)), or the result of very complex experimental procedures which even as laboratory processes are for all practical purposes limited to the production of compounds having molecular Weights of the order of 2,000 or less. Thus, the process of the present invention provides a novel class of synthetic polypetides having molecular weights sufficiently high so that self-supporting sheets, films, foils, fibers, filaments, ribbons, cast articles of predetermined other shapes, and the like can be formed therefrom; that is, synthetic polypeptide copolymers having molecular weights of the order of 13,000 or higher.

The properties which characterize the polypeptide copolymers of this invention adapt them to a variety of uses. They can be drawn into fibers useful as artificial silk, artificial hair bristles, threads, filaments, yarns, strips, films, bands,and the like. The materials of the present invention also can be employed to advantage in paints, varnishes, lacquers and enamels in several ways. They can be used as substitutes for the resin constituents, as substitutes for the softener constituent, as substitutes for the total resin constituent or the softener constituent in clear or pigmented lacquer compositions. These resins also can be used for coating sheet materials such as, cloth, paper and leather. They can be used in this respect in combination with other plastic high polymers or plastics such as pyroxylin and also in combination with softening ingredients such as castor oil, cottonseed oil, ortricresyl phosphate. They also can be used as modifying ingredients in the preparation of plastic compositions for use either in the fabrication of molded articles or sheeted materials for use in various applications such as in the manufacture of safety glass.

It is to be understood that in all these applications, the compositions disclosed in this invention can be used either alone or in combination with cellulose derivatives such as cellulose acetate, cellulose nitrate, ethyl cellulose and benzyl cellulose; in combination with natural resins such as rosin, damar, Congo, Pontianac and Manila gums; along with synthetic resins such as phenol-formaldehyde, urea-formaldehyde, and polyhydric alcohol-polybasic acid condensation products; and in combination with softeners such as triacetin, triphenyl phosphate, dibutyl phthalate, tricresyl phosphate, cellosolve 7 stearate, acet liaurih; as well as'castor oil, cott'on's'eed oil, and other vegetable oils.

The instant application is a continuation-inpart of my oopending application Serial No. 753,804, filed June 10, 1947', now abandoned.

Since certain changes can be made in thi'siirvention without departing from the spirit and Scope thereof, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limitmg sense.

I claim:

1. The process of preparing synthetic linear polype tide copol'ymers comprising thecofidensa: tion of a mixture of N'-'caihoxy-L-leucineanhydride and N-carboxy-Dkphenylalanine anhy= d'ride, with the evolution of carbon dioxide, man inert organic solvent and in the presence 'of trace amounts of an initiator having an active hydrogen atom, and said copolymers are char 'ac teriied by benzene isolubility, a degree'of pownlefization of at least 100', and fiber and filinfoiining ability.

2. The process of claim 1 wherein the reaction mixture consists of a substantially 1:1 molar ratio of 'the N-carboxy=anhydri'ds, the solvent is benzene, and the initiator is Water in an amount not exceeding about 0166 per cent by weight, based on the N-o'arboxy'anhydride matii're,

A linear polypeptidecopolymer of L-leuci'ne and nL-phenyialamne having the s'tr'iictil'ial formula:

H 'H, z-[r Lc-i il-n wherein R is selected from the group consistmg terial quantities, Z is thenon active hyiirogen moiety of the initiator, and 'n isan integer of at least 100, said copolymers being characterized by henzene 'solubility and fiber and film-forming ability. v I

e. A linear polypeptide 'copolymer of Llehcin'e andfDL-phenylalan'ine having the structii'i'ail formula:

benzene-solubility and fiber and film-forming ability. r

5. The linear polypeptide 'copolymer of claim a vvherein the copoly nei consists of a substantially l zl fnolar ratio of L-leucine and DL- phehylalanihe. I

The process of producing a polypeptide fihe'r comprising the extrusion of a benzene sp ntion of the copol'yiner of L-leucine and DL- phenylalanine, said copolymer having a degree 6i polymerization of at least 100, into 'a. medium which extracts the ber'izen'ea" d is-a non-solvent for said co polyiher. v t

'7. The 'fiher produced by the semen-hon sdivntspjmnm'g of a 'copolymer of L-leucine and Db-phenylalanine, yvhere'in "the said copblyine'i has a degree of polymerization of at least I00 and the ccpolyrher comprises a substantially equiinoleculai' quantity of the monomer @1118. ROBERT E. WOODWARD.

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1. THE PROCESS OF PREPARING SYNTHETIC LINEAR POLYPEPTIDE COPOLYMERS COMPRISING THE CONDENSATION OF A MIXTURE OF N-CARBOXY-L-LEUCINE ANHYDRIDE AND N-CARBOXY-DL-PHENYLALANINE ANHYDRIDE, WITH THE EVOLUTION OF CARBON DIOXIDE, IN AN INERT ORGANIC SOLVENT AND IN THE PRESENCE OF TRACE AMOUNTS OF AN INITIATOR HAVING AN ACTIVE HYDROGEN ATOM, AND SAID COPOLYMERS ARE CHARACTERIZED BY BENZENE-SOLUBILITY, A DEGREE OF POLYMERIZATION OF AT LEAST 100, AND FIBER AND FILMFORMING ABILITY. 